The development and maintenance of long-lived memory CD8 T cells are critical for effective vaccination and long-term immunity against re-infection. During viral infection, antigen specific nave CD8 T cells undergo rapid clonal expansion, followed by massive programmed contraction, leaving behind a population of memory precursor cells that mature into self-renewing memory CD8 T cells. The resulting memory cells form a layered defense system dependent on three distinct subsets of cells: tissue resident memory (TRM), effector memory (TEM), and central memory (TCM) CD8 T cells. TRM cells reside at mucosal and barrier surfaces, such as the intestinal tract, where infection is firs established and function as sentinels that rapidly recruit circulating TEM and TCM cells to the tissue upon pathogen re-exposure. Activated CD8 T cells are driven to effector or memory fates based in part on the level of signaling they receive through the PI3K/AKT pathway. While low PI3K/AKT signaling favors memory generation by enhancing expression and activity of stem-promoting factors and pro-survival genes including FoxO1, high PI3K/AKT signaling driven by TCR and inflammatory cytokine signaling promotes effector at the expense of memory cell formation. Two key regulators of T cell development, STAT3 and FoxO1, have recently been found to have important roles in the generation of protective memory cells. The importance of STAT3 in CD8 T cell memory formation has been corroborated in human patients with autosomal-dominant Hyper IgE Syndrome (HIES), a disease in which STAT3 is mutated to a dominant negative form. A central, but poorly understood, question in T cell biology is what signals and genes control the differentiation of TEM, TCM and TRM cells and how are their actions coordinated to provide optimal protection against infection? Our preliminary data suggest that the transcription factor STAT3 insulates CD8 T cells from inflammatory cytokine signaling by suppressing the PI3K/AKT pathway and that FoxO1 is the physiologically important downstream target. We hypothesize that STAT3 coordinates circulating and tissue-resident memory CD8 T cell differentiation by suppressing the PI3K/AKT pathway and enhancing the activity of FoxO1, a known regulator of memory defining genes importantly including TCF1/LEF1. Specific Aims: This proposal contains three specific aims. The first aim investigates the functional relationship between FoxO1 and STAT3 in both mouse and human memory CD8 T cells. In the second aim, the role of STAT3 as an insulator from inflammation will be investigated in mouse and human CD8 T cells. The third aim will investigate the role of STAT3 and FoxO1 in TRM formation. Relevance: Successful completion of these aims will expand our understanding of memory CD8 T cell differentiation and may identify a potential therapeutic intervention for human HIES patients with defective CD8 T cell immunity. Understanding the STAT3/FoxO1 axis is highly significant in T cell biology since this axis is important in TH17 and TReg formation. The STAT3/FoxO1 axis may represent an important transcriptional node in many different cell types.